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ABSTRACT

The present invention relates to pharmaceutical compositions comprising a positive modulator of a nicotinic receptor agonist, said positive modulator having the capability to increase the efficacy of the said nicotinic receptor agonist.

TECHNICAL FIELD

The present invention relates to pharmaceutical compositions comprisinga positive modulator of a nicotinic receptor agonist, said positivemodulator having the capability to increase the efficacy of the saidnicotinic receptor agonist.

BACKGROUND ART

Cholinergic receptors normally bind the endogenous neurotransmitteracetylcholine (ACh), thereby triggering the opening of ion channels. AChreceptors in the mammalian central nervous system can be divided intomuscarinic (mAChR) and nicotinic (nAChR) subtypes based on the agonistactivities of muscarine and nicotine, respectively. The nicotinicacetylcholine receptors are ligand-gated ion-channels containing fivesubunits (for reviews, see Colquhon et al. (1997) Advances inPharmacology 39, 191-220; Williams et al. (1994) Drug News &Perspectives 7, 205-223; Doherty et al. (1995) Annual reports inMedicinal Chemistry 30, 41-50). Members of the nAChR gene family havebeen divided into two groups based on their sequences; members of onegroup are considered β subunits, while a second group are classified asα subunits (for reviews, see Karlin & Akabas (1995) Neuron 15,1231-1244; Sargent (1993) Annu. Rev. Neurosci. 16, 403-443). Three ofthe α subunits, α7, α8 and α9, form functional receptors when expressedalone and thus presumably form homooligomeric receptors.

An allosteric transition state model of the nAChR involves at least aresting state, an activated state and a “desensitized” closed channelstate (Williams et al., supra; Karlin & Akabas, supra). Different nAChRligands can thus differentially stabilize the conformational state towhich they preferentially bind. For example, the agonists ACh and(−)-nicotine stabilize the active and desensitized states.

Changes of the activity of nicotinic receptors has been implicated in anumber of diseases. Some of these, e.g. myasthenia gravis and ADNFLE(autosomal dominant nocturnal front lobe epilepsy) (Kuryatov et al.(1997) J. Neurosci. 17(23):9035-47), are associated with reductions inthe activity of nicotinic transmission either through a decrease inreceptor number or increased desensitization, a process by whichreceptors become insensitive to the agonist. Reductions in nicotinicreceptors have also been hypothesized to mediate cognitive deficits seenin diseases such as Alzheimer's disease and schizophrenia (Williams etal., supra). The effects of nicotine from tobacco are also mediated bynicotinic receptors. Increased activity of nicotinic receptors mayreduce the desire to smoke.

However, treatment with nicotinic receptor agonists which act at thesame site as ACh is problematic because ACh not only activates, but alsoblocks receptor activity through processes which include desensitization(for a review, see Ochoa et al. (1989) Cellular and MolecularNeurobiology 9, 141-178) and uncompetitive blockade (open-channel block)(Forman & Miller (1988) Biophysical Journal 54(1):149-58). Furthermore,prolonged activation appears to induce a long-lasting inactivation.Therefore agonists of ACh can be expected to reduce activity as well asenhance it. At nicotinic receptors in general, and, of particular note,at the α7-nicotinic receptor, desensitization limits the duration ofcurrent during agonist application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

Model of current traces elicited by agonist, representing determinationof increase in agonist efficacy by determination of current amplitude.Bars denote duration of application of compounds.

FIG. 2

Model of current traces elicited by agonist, representing determinationof increase in agonist efficacy by determination of “area under thecurve”. Arrow indicates overlay of ACh current and ACh+modulatorcurrent. Bars denote duration of application of compounds.

FIG. 3

Effect of 5-hydroxyindole on ACh activity on the α7-nicotinic receptor.The current value of 100% is the extrapolated maximum from the AChcurve.

() ACh

(∘) ACh+0.5 mM 5-hydroxyindole

FIG. 4

Effect of 5-OHi on “area under the curve” for saturating concentrationof agonist.

FIG. 5

Effect of 5-hydroxyindole on ACh (open staples) and AR-R-17779 (filledstaples) activity on the α7-nicotinic receptor.

FIG. 6

Effect of nAChR α7 modulator on agonist activity as measured by Ca²⁺flux through nAChR α7 expressed in HEK-293 cells. Filled box is signalobtained in the absence of agonist and the absence of modulator. Filledstaples is signal obtained in the presence of agonist alone (nomodulator). Open box is signal obtained in the presence of agonist andmodulator.

DISCLOSURE OF THE INVENTION

It has surprisingly been found that certain compounds, e.g.5-hydroxyindole (5-OHi), can enhance the efficacy of agonists atnicotinic receptors. This increase in efficacy can be greater than2-fold. It is believed that compounds having this type of action(hereinafter referred to as “positive modulators”) will be particularlyuseful for treatment of conditions associated with reductions innicotinic transmission. In a therapeutic setting such compounds couldrestore normal interneuronal communication without affecting thetemporal profile of activation. In addition, they would not producelong-term inactivation as prolonged application of agonist may.

The presence of this efficacy enhancing activity could not be predictedby the prior art. Albuquerque et al. have reported on another allostericsite on nicotinic receptors, which they call a “noncompetitive agonist”site. Compounds acting at this site are also called “allostericallypotentiating ligands” (APL's). Compounds which appear to act at thissite include several cholinesterase inhibitors, codeine, and 5-HT. Ithas been stated that activity via this noncompetitive agonist site “doesnot affect the level of maximum response to ACh; it shifts thedose-response curve to the left” (Maelicke & Albuquerque (1996) DDT,vol. 1, 53-59). In specific distinction, compounds acting at thediscovered site increase the maximum response to ACh (its efficacy).

Another distinction between APL's and the present invention is theeffect they have on total current (as measured by area under the curve)in the presence of a saturating concentration of agonist. APL's havelittle to no effect on area under the curve on nAChR α7 expressed inoocytes; 8-10% increases in area under the curve for a 1 second agonistapplication have been observed. In contrast, 5-OHi causes a robustincrease in area under the curve (˜400% increase) under the sameconditions (see FIG. 4).

Specificity of the effect within the nicotinic receptor family is yetanother distinguishing characteristic between APL's and the invention.APL's exert there positive modulatory effect on all nicotinic receptorstested, including muscle type (α1βδe), whereas no positive modulatoryeffect has been observed with 5-OHi at either of these receptorsubtypes.

At some non-nicotinic receptors, compounds have been found which candecrease receptor desensitization. At AMPA-type excitatory amino acidreceptors, compounds such as cyclothiazide, some lectins like wheat-germagglutinin, piracetam-like nootropics, and AMPAkines have been shown todecrease desensitization (Partin et al. (1993) Neuron 11, 1069-1082).Glycine has been reported to reduce desensitization of NMDA-typeexcitatory amino acid receptors (Mayer et al. (1989) Nature 338,425-427). However, compounds which decrease desensitization on onereceptor group have been found, in general, not to have the same affecton other receptor groups. For instance cyclothiazide has little or noeffect on the NMDA and KA subtypes of glutamate receptors (Partin et al.(1993) Neuron 11, 1069-1082); moreover cyclothiazide is found to block5-HT₃ receptors (D. A. Gurley, unpublished results). Glycine has noeffect on 5-HT₃ receptors (Gurley and Lanthom, (in press) Neurosci.Lett.).

The site was discovered using a compound (5-OHi) which is known todecrease desensitization at the 5-HT₃ receptor (Kooyman. A. R. et al.(1993) British Journal of Pharmacology 108, 287-289). However, only oneother compound which produces or increases activity at the 5-HT₃receptor, 5-HT itself, has been reported to increase activity atnicotinic receptors (Schrattenholz et al. (1996) Molecular Pharmacology49, 1-6) although this activity has never been reported in Xenopusoocytes. Most agonists at the 5-HT₃ receptor have no activity or areantagonists at nicotinic receptors (unpublished results). In addition,the present inventors have been unable to reproduce the finding that5-HT increases activity at a nicotinic receptor. Therefore the enhancingeffect of 5-OHi at nicotinic receptors could not have been predicted.

Consequently, the present invention provides in a first aspect to apharmaceutical composition comprising a positive modulator of anicotinic receptor agonist together with a pharmaceutically acceptablecarrier. For the purposes of the present invention, the term “positivemodulator” or “positive modulator of a nicotinic receptor agonist” shallbe understood as a compound having the capability to increase themaximum efficacy of a nicotinic receptor agonist.

It will be understood that the invention includes compositionscomprising either a positive modulator as the only active substance,thus modulating the activity of endogenous nicotinic receptor agonists,or a positive modulator in combination with a nicotinic receptoragonist.

In a preferred form of the invention, the said positive modulator is5-hydroxyindole.

In another preferred form of the invention, the said nicotinic receptoragonist is an α7-nicotinic receptor agonist. Examples of α7-nicotinicreceptor agonists are known in the art, e.g. from WO 96/06098 and WO97/30998.

In a further aspect, the invention provides a method for the treatmentof a condition associated with reduced nicotine transmission, byadministering to a patient in need of such treatment, a medicallyeffective amount of a positive modulator of a nicotinic receptoragonist, said positive modulator having the capability to increase theefficacy of the said nicotinic receptor agonist.

It will be understood that the said positive modulator can beadministered either with the purpose of acting on endogenous nicotinereceptor agonists, or in combination with an exogenous nicotinicreceptor agonist.

In a further aspect, the invention provides a method for identifying apositive modulator of a nicotinic receptor agonist. Compounds areconsidered “positive modulators” if, in the presence of saturatingconcentrations of the nAChR α7 agonist ACh, current is elicited thatexceeds 200% of control current (100% potentiation) when measuredbaseline to peak (see Experimental Methods). Control current is definedas the current elicited by agonist in the absence of modulator. Asaturating concentration of ACh is defined as 10-times the EC₅₀ for thespecific nAChR α7 type used. EC₅₀ is defined as the concentration whichelicits a half-maximal response. EC₅₀ values for nAChR α7 subtypestypically range between 100-300 μM (Bertrand et al. (1992) NeuroscienceLetters 146, 87-90; Peng et al. (1994) Molecular Pharmacology 45,546-554). Further, compounds are considered “positive modulators” if, inthe presence of saturating concentrations of agonist, total currentthrough the receptor (flux) exceeds 200% of control current. One measureof total current is area under the curve (current trace) during anagonist application.

Consequently, the method according to the invention for identifying apositive modulator of a nicotinic receptor agonist, can comprise thesteps (a) expressing a nicotinic receptor on the surface of a cell; (b)contacting the said nicotinic receptor with a compound known to be anicotinic receptor agonist and a compound to be tested for positivemodulating activity; (c) determining whether the compound to be testedexhibits a positive modulation on the effect of the said nicotinicreceptor agonist resulting in current amplitude (measured baseline topeak) or total current (measured as area under the curve for the currenttrace) greater than 200% of control (100% potentiation).

In yet a further aspect, the invention provides a method for identifyinga compound which is a nicotinic receptor agonist, said method comprisingthe steps (a) expressing a nicotinic receptor on the surface of a cell;(b) contacting the said nicotinic receptor with a compound to be testedfor nicotinic receptor agonist activity, in the presence of a positivemodulator of a nicotinic receptor agonist; and (c) determining whetherthe compound to be tested exhibits nicotinic receptor agonist activity.It will be understood by the skilled person that “nicotinic receptoragonist activity” can be determined by methods known in the art, such asthose methods described in the section “Experimental Methods” below.

Experimental Methods

(a) Xenopus Oocyte Current Recording

The Xenopus oocyte has provided a powerful means of assessing thefunction of proteins thought to be subunits of ligand-gatedion-channels. Injection of RNA transcribed from cDNA clones encoding theappropriate receptor subunits, or injection of cDNA in which the codingsequence is placed downstream of a promoter, results in the appearanceof functional ligand-gated ion-channels on the surface of the oocyte(see e.g. Boulter et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84,7763-7767).

Consequently, one convenient technique to assess the enhancement ofnicotinic efficacy is two-electrode voltage-clamp recording from Xenopusoocytes expressing α7-nicotinic receptors from human cRNA.

Xenopus laevis frogs (Xenopus I, Kalamazoo, Mich.) were anesthetizedusing 0.15% tricaine. Oocytes were removed to OR2 solution (82 mM NaCl,2.5 mM KCl, 5 mM HEPES, 1.5 mM NaH₂PO₄, 1 mM MgCl₂, 0.1 mM EDTA; pH7.4). The oocytes were defolliculated by incubation in 25 ml OR2containing 0.2% collagenase 1 A (Sigma) two times for 60 min on aplatform vibrating at 1 Hz and stored in Leibovitz's L-15 medium (50μg/ml gentomycin, 10 Units/ml penicillin, and 10 μg/ml streptomycin).Approximately 50 ng of cRNA was injected in each oocyte the followingday. cRNA was synthesised from cDNA using Message Machine (purchasedfrom Abion).

The external recording solution consisted of 90 mM NaCl, 1 mM KCl, 1 mMMgCl₂, 1 BaCl₂, 5 mM HEPES; pH 7.4. Two-electrode voltage-clamprecording was carried out using an Oocyte Clamp amplifier (OC 725C;Warner Instrument, Hamden, Conn.). Oocytes were impaled with twoelectrodes of 1-2 MΩ tip resistance when filled with 3M KCl. Recordingswere begun when membrane potential became stable at potentials negativeto −20 mV (resting membrane potentials are less negative when Ba⁺⁺replaces Ca⁺⁺ in bathing solutions). Membrane potential was clamped at−80 mV. ACh was purchased from Sigma. Oocytes were continuously perfused(5 ml/min) with recording solution with or without ACh.

Current amplitude was measured from baseline to peak. EC₅₀ values,maximal effect, and Hill slopes were estimated by fitting the data tothe logistic equation using GraphPad Prism (GraphPad Software, Inc., SanDiego, Calif.).

Increases in agonist efficacy elicited by a positive modulator can becalculated in two ways:

(1) As percent potentiation of current amplitude which is defined as100(I_(m)-I_(c))/I_(c) where I_(m) is current amplitude in the presenceof modulator and I_(c) is current in the absence of modulator (FIG. 1).

(2) As percent potentiation of “area under curve” of an agonist trace.Area under the curve is a common representation of the total ion fluxthrough the channel (FIG. 2). In the example shown in FIG. 2, althoughcurrent amplitude is not increased, area under the curve is potentiatedroughly 100% over control for the duration of the agonist application

(b) Ca²⁺ Flux Imaging

Imaging of Ca²⁺ flux through nAChR α7 receptors transiently expressed ina cell line is another means of assaying modulator activity.

Cells expressing α7 receptors are grown to confluence in 96 well platesand loaded with fluo-3, a fluorescent calcium indicator. To screen forα7 modulatory activity, the 96 well plate is placed in a fluorescenceimaging plate reader (FLIPR) and test compounds along with an α7 agonistare applied simultaneously to all wells. Receptor activation is measuredby calcium influx into cells which is quantified by the increase influorescence intensity of each well, recorded simultaneously by theFLIPR. Amodulatory effect is determined by the increase in fluorescenceover that of agonist alone. Similarly, to test for nAChR α7 agonistactivity, test compounds along with an α7 modulator are appliedsimultaneously to all wells. Receptor activation is measured by calciuminflux into cells which is quantified by the increase in fluorescenceintensity of each well, recorded simultaneously by the FLIPR. An agonisteffect is determined by the increase in fluorescence over that ofmodulator alone.

EXAMPLE 1

Changes in efficacy of nicotinic agonists was assessed by measuring thecombined effects of a nicotinic agonist with test compounds. In general,the protocol consisted of pretreatment with test compound pluscoapplication of agonist and test compound. 5-hydroxyindole was testedat 500 μM against a range of concentrations of ACh. ACh was first testedby itself so that an EC₅₀ and maximal response could be determined. Thenthe same concentrations of ACh were applied along with 5-OH-indole. Theresults (FIG. 3) were that the maximal response to ACh was increased(maximum amplitude increased 2-fold).

The effect of 5-OHi (0.5 mM) on “area under curve” for saturatingconcentration of agonist (3 mM ACh) was determined. 5-OHi caused arobust increase in area under the curve (˜400% increase) (FIG. 4).

Applied by itself, 5-hydroxyindole did not induce current in oocytesinjected with cRNA for wild-type α7 nicotinic receptors.

EXAMPLE 2

The effect of 5-hydroxyindole on various nicotinic agonists was tested.The increase in efficacy afforded by 5-hydroxyindole was seen with allnicotinic agonists tested, e.g.AR-R 17779 (FIG. 5). Open boxes currentelicited by ACh (3 mM) with (+) and without (−) modulator. Solid boxescurrent elicited by a nicotinic agonist designated AR-R 17779 (100 μM)with (+) and without (−) modulator. Modulator in this instance was 1 mM5-OHi.

Compounds tested with similar results include (−)-nicotine and choline(data not shown). Therefore the effect appears to be general for anycholinergic agonist.

EXAMPLE 3

The increase in efficacy afforded by 5-hydroxyindole was not seen on anyother nicotinic receptors, e.g. mouse muscle-type nicotinic receptors.

EXAMPLE 4

A series of related compounds were tested for positive modulation on AChactivity. Only a few compounds retained efficacy enhancing activity. Inparticular, serotonin (5-HT) did not increase efficacy. This preliminaryanalysis of close analogues indicates a fairly tight structure-activityrelationship, suggesting a selective site of action.

EXAMPLE 6

Effect of nAChR α7 modulator on agonist activity was measured by Ca²⁺flux through nAChR α7 expressed in HEK-293 cells. The nicotinic agonistdesignated AR-R-17779 was used. The results are shown in FIG. 6. Nodiscernible signal was obtained in the presence of agonist alone (nomodulator). In the presence of agonist together with modulator, asignificant increase in agonist activity was seen.

What is claimed is:
 1. A method for identifying an efficacy enhancer ofan angonist of an α7-nicotinic receptor, said method comprising thesteps of: (a) expressing said nicotinic receptor on the surface of acell; (b) contacting said nicotinic receptor with a compound known to bea nicotinic receptor agonist and a compound to be tested for efficacyenhancement activity; and, (c) determining whether said compound to betested exhibits enhancement of an effect of said nicotinic receptoragonist.
 2. A method for identifying a compound which is an agonist ofan α7-nicotinic receptor, said method comprising the steps of: (a)expressing said nicotinic receptor on the surface of a cell; (b)contacting said nicotinic receptor with a compound to be testednicotinic receptor agonist activity, in the presence of an efficacyenhancer of a nicotinic receptor agonist; and, (c) determining whethersaid compound to be tested exhibits nicotinic receptor agonist activity.